Preparation method of amino acid-based polypeptide hydrogel for promoting skin repair

Abstract

The invention discloses a preparation method of amino acid-based polypeptide hydrogel for promoting skin repair, and relates to the technical field of preparation of amino acid-based polypeptide hydrogel, the method comprises the following steps: S1, polypeptide design and synthesis; s2, carrying out a cross-linking reaction and initiating gelation; s3, washing and post-processing; s4, performing functional modification and sterilization treatment, wherein an amino acid sequence with self-assembly capability is selected in the step S1 (for example, a self-assembly amphiphilic short peptide S1 (AEAEAKAKAEAEAKAK) or Gly-Ile-Trp-Leu-Gly is taken as a starting sequence). According to the preparation method of the amino acid-based polypeptide hydrogel for promoting skin repair, a proper amount of cross-linking agent is added into the polypeptide solution, and the cross-linking agent can promote cross-linking reaction among polypeptides so as to form the hydrogel with a three-dimensional network structure, so that the biological activity and stability of the hydrogel can be enhanced, and the hydrogel can be used for repairing skin. More functional characteristics can be endowed, so that the requirements of different skin repairing scenes are met.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of amino acid-based polypeptide hydrogels, and in particular to a method for preparing amino acid-based polypeptide hydrogels for promoting skin repair. Background Art

[0002] As the largest organ in the human body, the skin not only protects internal tissues from external damage, but also participates in a variety of physiological activities such as temperature regulation and sensory conduction. However, after being damaged by trauma, burns, infection, etc., the skin has limited self-repair ability, and the repair process is often accompanied by adverse reactions such as scar formation and pigmentation. Therefore, the development of effective skin repair materials is of great significance to improving the quality of life of patients and reducing the medical burden. Peptide hydrogel is a three-dimensional network structure material based on polypeptides and formed by physical or chemical cross-linking. This material can not only simulate the microenvironment of the extracellular matrix of the skin and provide a platform for cells to attach and grow, but also regulate cell proliferation, differentiation and migration through its unique physical and chemical properties, thereby promoting skin repair.

[0003] For example, the Chinese patent with the announcement number CN110028552A discloses a method for preparing a self-assembling polypeptide and its hydrogel, which belongs to the field of biomaterial technology. The self-assembling polypeptide of the present invention has an amino acid sequence of GlyIle-Trp-Leu-Gly. The present invention breaks through the existing research ideas and methods of polypeptide hydrogels at home and abroad, and the length of the designed and synthesized polypeptide sequence is short; the preparation method of the polypeptide hydrogel is simple and rapid; the biocompatibility of the prepared polypeptide hydrogel is good. The present invention provides flexibility for the chemical design of the preparation of peptide-based hydrogels, provides simplicity for polypeptide synthesis, and contributes to the development and utilization of peptide-based hydrogel materials.

[0004] The existing technology has the following problems:

[0005] The design of the existing preparation method of amino acid-based polypeptide hydrogel is to modify the original self-assembling polypeptide system. It lacks the selection of amino acid sequences with self-assembly ability, and its design is limited. At the same time, the skin repair process of the existing prepared hydrogel is slow. Therefore, it is necessary and meaningful to develop a new self-assembling polypeptide system. Summary of the invention

[0006] The present invention provides a method for preparing an amino acid-based polypeptide hydrogel for promoting skin repair, so as to solve the problems raised in the above-mentioned background technology.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] A method for preparing an amino acid-based polypeptide hydrogel for promoting skin repair, the method comprising the following steps:

[0009] S1. Peptide design and synthesis;

[0010] S2, cross-linking reaction and initiation of gelation;

[0011] S3, washing and post-processing;

[0012] S4. Functional modification and sterilization treatment.

[0013] Preferably, in step S1, an amino acid sequence with self-assembly ability is selected (such as taking the self-assembly amphiphilic short peptide S1 (AEAEAKAKAEAEAKAK) or GlyIle-Trp-Leu-Gly as the starting sequence); these sequences usually contain amino acids that can form hydrogen bonds, hydrophobic interactions or ionic interactions, such as arginine, glycine, phenylalanine, etc.;

[0014] Dissolve the selected polypeptide in an appropriate amount of deionized water, and adjust the pH value of the solution to a range suitable for the reaction (usually weakly acidic or neutral, depending on the properties of the selected amino acid).

[0015] Preferably, the concentration of the polypeptide in step S1 can be adjusted as needed and set between 1% and 10% (w / v), and the concentration range of the polypeptide solution is mostly selected to be 5.5 to 14.69 mM.

[0016] Preferably, in step S2, a suitable amount of cross-linking agent (such as EDC / NHS) is added to the polypeptide solution. These agents can promote cross-linking reactions between polypeptides, thereby forming a hydrogel with a three-dimensional network structure.

[0017] Preferably, the cross-linking reaction in step S2 is carried out under stirring for 30 minutes to 2 hours to ensure that the cross-linking agent is evenly distributed and fully reacted.

[0018] Preferably, in step S2, a suitable amount of initiator or catalyst (such as APS and TEMED) is added, and these reagents can promote the rapid formation of gel;

[0019] After mixing evenly, pour the solution into a mold and let it stand for a period of time to completely gel (usually several hours to a day, the specific time depends on the reaction speed of the system).

[0020] Preferably, after the synthesis reaction of the hydrogel is completed in step S3, the formed hydrogel is taken out from the mold and washed multiple times with distilled water or deionized water to remove impurities such as unreacted monomers, catalysts, solvents, etc., and the temperature is set at 4° C.;

[0021] The washing process can be performed by soaking and changing water for multiple times, and the setting time is generally between 24 and 48 hours; the pH value of the washing solution is set to 3-10.

[0022] Preferably, in step S4, in order to further enhance the skin repair effect of the hydrogel, biomolecule binding can be introduced after gelation: growth factors (such as VEGF, PDGF) can be combined with polypeptide chains to promote the proliferation, differentiation and angiogenesis of skin cells and accelerate the skin repair process;

[0023] Finally, to ensure the safety of the hydrogel in biological applications, it can be sterilized using ultraviolet light (high temperature and high pressure sterilization or filtration sterilization, etc. can also be used) so that it is suitable for later skin repair experiments or clinical applications.

[0024] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0025] 1. The present invention provides a method for preparing an amino acid-based polypeptide hydrogel that promotes skin repair. By adding an appropriate amount of cross-linking agent to the polypeptide solution, these agents can promote a cross-linking reaction between polypeptides, thereby forming a hydrogel with a three-dimensional network structure, which can not only enhance the biological activity and stability of the hydrogel, but also give it more functional properties to meet the needs of different skin repair scenarios.

[0026] 2. The present invention provides a method for preparing an amino acid-based polypeptide hydrogel that promotes skin repair. When preparing the amino acid-based polypeptide hydrogel that promotes skin repair, selecting an amino acid sequence with self-assembly ability is a key step. The design of these sequences should be based on a deep understanding of their self-assembly mechanism and biological activity, thereby improving effectiveness and safety in practical applications.

[0027] 3. The present invention provides a method for preparing an amino acid-based polypeptide hydrogel that promotes skin repair. By introducing biological molecules after gelation, growth factors are combined with polypeptide chains to promote skin cell proliferation, differentiation and angiogenesis, thereby accelerating the skin repair process. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention is a schematic diagram of the process structure of the method for preparing the amino acid-based polypeptide hydrogel for promoting skin repair. DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0030] like Figure 1As shown, a method for preparing an amino acid-based polypeptide hydrogel for promoting skin repair comprises the following steps:

[0031] S1. Peptide design and synthesis;

[0032] S2, cross-linking reaction and initiation of gelation;

[0033] S3, washing and post-processing;

[0034] S4. Functional modification and sterilization treatment.

[0035] like Figure 1 As shown, in step S1, an amino acid sequence with self-assembly ability is selected (such as taking the self-assembly amphiphilic short peptide S1 (AEAEAKAKAEAEAKAK) or GlyIle-Trp-Leu-Gly as the starting sequence); these sequences usually contain amino acids that can form hydrogen bonds, hydrophobic interactions or ionic interactions, such as arginine, glycine, phenylalanine, etc.;

[0036] The selected polypeptide is dissolved in an appropriate amount of deionized water, and the pH value of the solution is adjusted to a range suitable for the reaction (usually weakly acidic or neutral, depending on the properties of the selected amino acid).

[0037] like Figure 1 As shown, the concentration of the polypeptide in step S1 can be adjusted as needed and set between 1% and 10% (w / v). Most of the selected polypeptide solution concentrations are in the range of 5.5 to 14.69 mM.

[0038] like Figure 1 As shown, in step S2, a proper amount of cross-linking agent (such as EDC / NHS) is added to the polypeptide solution. These agents can promote cross-linking reactions between polypeptides, thereby forming a hydrogel with a three-dimensional network structure.

[0039] like Figure 1 As shown, the cross-linking reaction in step S2 is carried out under stirring conditions for 30 minutes to 2 hours to ensure that the cross-linking agent is evenly distributed and fully reacted.

[0040] like Figure 1 As shown, in step S2, a proper amount of initiator or catalyst (such as APS and TEMED) is added, and these reagents can promote the rapid formation of gel;

[0041] After mixing evenly, pour the solution into a mold and let it stand for a period of time to completely gel (usually several hours to a day, the specific time depends on the reaction speed of the system).

[0042] like Figure 1As shown, in step S3, after the synthesis reaction of the hydrogel is completed, the formed hydrogel is taken out from the mold and washed multiple times with distilled water or deionized water to remove impurities such as unreacted monomers, catalysts, solvents, etc., and the temperature is set at 4°C;

[0043] The washing process can be carried out by soaking and changing the water several times, and the setting time is generally between 24 and 48 hours; the pH value of the washing solution is made to be 3-10, and the pH value of the washing solution is 7 optimally.

[0044] like Figure 1 As shown, in step S4, in order to further enhance the skin repair effect of the hydrogel, biomolecule binding can be introduced after gelation: growth factors (such as VEGF, PDGF) are combined with polypeptide chains to promote the proliferation, differentiation and angiogenesis of skin cells and accelerate the skin repair process;

[0045] Finally, to ensure the safety of the hydrogel in biological applications, ultraviolet sterilization (high temperature and high pressure sterilization or filtration sterilization, etc.) is required to sterilize it so that it is suitable for later skin repair experiments or clinical applications.

[0046] The working principle of the present invention is as follows: by selecting amino acid sequences with self-assembly ability; these sequences usually contain amino acids that can form hydrogen bonds, hydrophobic interactions or ionic interactions, such as arginine, glycine, phenylalanine, etc., dissolving the selected polypeptide in an appropriate amount of deionized water, and the concentration of the polypeptide can be adjusted as needed and set between 1% and 10% (w / v), and the concentration range of the polypeptide solution is mostly selected to be 5.5-14.69mM; adding an appropriate amount of cross-linking agent (such as EDC / NHS) to the polypeptide solution, these reagents can promote cross-linking reactions between polypeptides, thereby forming a hydrogel with a three-dimensional network structure, and the cross-linking reaction is under stirring conditions, and the reaction time is between 30 minutes and 2 hours to ensure that the cross-linking agent is evenly distributed and fully reacted; adding an appropriate amount of initiator or catalyst (such as APS and TEMED), these reagents can promote the rapid formation of gel; after mixing evenly, pouring the solution into a mold. , let it stand for a period of time to completely gel (usually several hours to a day, the specific time depends on the reaction speed of the system). After the synthesis reaction of the hydrogel is completed, the formed hydrogel is taken out of the mold and washed several times with distilled water or deionized water to remove unreacted monomers, catalysts, solvents and other impurities. The temperature is set at 4°C; the washing process can be carried out by soaking and changing water multiple times, and the setting time is generally between 24 and 48 hours; the pH value of the washing solution is set to 7; in order to further enhance the skin repair effect of the hydrogel, biological molecules can be introduced after gelation: growth factors (such as VEGF, PDGF) are combined with polypeptide chains to promote the proliferation, differentiation and angiogenesis of skin cells and accelerate the skin repair process; finally, in order to ensure the safety of the hydrogel in biological applications, ultraviolet sterilization (high temperature and high pressure sterilization or filtration sterilization, etc.) needs to be sterilized.

[0047] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing an amino acid-based polypeptide hydrogel for promoting skin repair, characterized in that: The method comprises the following steps: S1. Peptide design and synthesis; S2, cross-linking reaction and initiation of gelation; S3, washing and post-processing; S4. Functional modification and sterilization treatment.

2. The method for preparing an amino acid-based polypeptide hydrogel for promoting skin repair according to claim 1, characterized in that: In step S1, an amino acid sequence having self-assembly ability is selected; these sequences usually contain amino acids capable of forming hydrogen bonds, hydrophobic interactions or ionic interactions, such as arginine, glycine, phenylalanine, etc.; Dissolve the selected polypeptide in an appropriate amount of deionized water and adjust the pH value of the solution to a range suitable for the reaction.

3. The method for preparing an amino acid-based polypeptide hydrogel for promoting skin repair according to claim 2, characterized in that: The concentration of the polypeptide in step S1 can be adjusted as needed and set between 1% and 10% w / v. The concentration range of the polypeptide solution is usually 5.5 to 14.69 mM.

4. The method for preparing an amino acid-based polypeptide hydrogel for promoting skin repair according to claim 1, characterized in that: In step S2, a proper amount of cross-linking agents is added to the polypeptide solution. These agents can promote cross-linking reactions between polypeptides, thereby forming a hydrogel with a three-dimensional network structure.

5. The method for preparing an amino acid-based polypeptide hydrogel for promoting skin repair according to claim 4, characterized in that: The cross-linking reaction in step S2 is carried out under stirring conditions for 30 minutes to 2 hours to ensure that the cross-linking agent is evenly distributed and fully reacted.

6. The method for preparing an amino acid-based polypeptide hydrogel for promoting skin repair according to claim 5, characterized in that: In step S2, a suitable amount of initiator or catalyst is added, and these reagents can promote the rapid formation of gel; After mixing evenly, pour the solution into the mold and let it stand for a while to completely gel.

7. The method for preparing an amino acid-based polypeptide hydrogel for promoting skin repair according to claim 1, characterized in that: In step S3, after the synthesis reaction of the hydrogel is completed, the formed hydrogel is taken out from the mold and washed multiple times with distilled water or deionized water to remove impurities such as unreacted monomers, catalysts, solvents, etc., and the temperature is set at 4° C.; The washing process can be performed by soaking and changing water for multiple times, and the setting time is generally between 24 and 48 hours; the pH value of the washing solution is set to 3-10.

8. The method for preparing an amino acid-based polypeptide hydrogel for promoting skin repair according to claim 1, characterized in that: In step S4, in order to further enhance the skin repair effect of the hydrogel, biomolecule binding can be introduced after gelation: growth factors are combined with polypeptide chains to promote the proliferation, differentiation and angiogenesis of skin cells and accelerate the skin repair process; Finally, to ensure the safety of the hydrogel in biological applications, it can be sterilized using ultraviolet sterilization, making it suitable for later skin repair experiments or clinical applications.

Images